J Periodont Res 2010; 45: 129135 All rights reserved

2009 The Authors. Journal compilation 2009 Blackwell Munksgaard JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/j.1600-0765.2009.01212.x

Effects of topical application of lipopolysaccharide and proteases on hepatic injury induced by high-cholesterol diet in rats
Yamamoto T, Tomofuji T, Tamaki N, Ekuni D, Azuma T, Sanbe T. Eects of topical application of lipopolysaccharide and proteases on hepatic injury induced by high-cholesterol diet in rats. J Periodont Res 2010; 45: 129135. 2009 The Authors. Journal compilation 2009 Blackwell Munksgaard Background and Objective: Topical application of lipopolysaccharide and proteases to the gingival sulcus induced not only periodontal inammation but also detectable liver changes in rats fed a normal diet. However, these changes in the liver were not sucient to induce pathological consequences. The purpose of the present study was to investigate whether gingival inammation-induced liver change would have more dramatic pathological consequences in rats fed a high-cholesterol diet compared with the eect of the high-cholesterol diet alone. Material and Methods: Twenty-four male Wistar rats were divided into four groups. During an 8 week experimental period, two groups were fed a normal diet and the other two were fed a high-cholesterol diet containing 1% cholesterol (w/w) and 0.5% cholic acid (w/w). Four weeks prior to the end of the experimental period, one of each of the dietary groups received daily topical application of lipopolysaccharide and proteases to the gingival sulcus, while the other was treated with pyrogen-free water. Results: In the rats without application of lipopolysaccharide and proteases, the serum level of hexanoyl-lysine, scores of steatosis and inammation, and concentration of 8-hydroxydeoxyguanosine in liver of rats fed a high-cholesterol diet were higher than in those fed a normal diet. In rats fed a high-cholesterol diet, the scores of steatosis and inammation and the concentration of 8-hydroxydeoxyguanosine in the liver of rats with application of lipopolysaccharide and proteases were higher than in those without. Conclusion: In a rat model, application of lipopolysaccharide and proteases to the gingival sulcus augmented the eect of a high-cholesterol diet on steatosis, inammation and oxidative damage in the liver.

T. Yamamoto, T. Tomofuji, N. Tamaki, D. Ekuni, T. Azuma, T. Sanbe

Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikatacho, Okayama, Japan

Tatsuo Yamamoto PhD, DDS, Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Tel: +81 86 235 6711 Fax: +81 86 235 6714 e-mail: tatsuo@md.okayama-u.ac.jp Key words: periodontal disease; animal model; high-cholesterol diet; liver injury; oxidative stress Accepted for publication January 18, 2009

Periodontitis is the inammation of supporting structures of the tooth caused by chronic bacterial infection (1). Studies have suggested that periodontitis is a risk factor for systemic

diseases, including diabetes mellitus (2), hyperlipidemia (3) and coronary heart diseases (4). The mechanisms by which periodontitis increases the likelihood of these systemic diseases have

not been clearly dened, but the prerequisite is believed to be host response to long-term systemic exposure to bacterial pathogens [i.e. lipopolysaccharide (LPS) and proteases]. In spite

130

Yamamoto et al. induce functional disorder of the liver in rats (6), it may augment liver injury induced by a high-cholesterol diet because systemic oxidative stress is increased by application of a combination of LPS and proteases to the gingival sulcus (12). The purpose of the present study was to investigate the eects of topical application of a combination of LPS and proteases to the gingival sulcus on hepatic pathological changes and serum parameters of liver function in rats fed a high-cholesterol diet. In addition, since one of the common hepatic cellular responses to periodontal inammation and cholesterol overload is induction of free radical production (6,13), the changes in hepatic oxidative damage were also examined. (0.5 lL, three doses) and proteases (0.5 lL, three doses) or pyrogen-free water (0.5 lL, six doses) were introduced once a day using a micropipette into the gingival sulcus of both maxillary rst molars. The tip of the micropipette was placed close to the gingival sulcus, and 0.5 lL of the solutions (LPS and proteases, or pyrogen-free water) was dropped into the sulcus (14), within 1 h after induction of general anesthesia by inhalation of 24% isourane delivered in a O2 gas through a face mask.
Blood collection and measurement of biochemical markers

of numerous reports on the relationship between periodontitis and systemic diseases, little attention has been paid to the eects of periodontal infection on the liver, which plays an important role in glucose and lipid metabolism. Epidemiological studies demonstrated an association between periodontitis and liver diseases by showing that the incidence of periodontitis increased with elevated serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and cholinesterase, and an AST-to-ALT ratio of less than one (5). In our previous study (6), chronic application of LPS and proteases to the gingival sulcus induced non-alcoholic fatty liver disease-like lesion in rats, with increasing periodontal inammation and serum level of oxidative stress. The study also suggested that systemic oxidative stress, induced by application of a combination of LPS and proteases to the gingival sulcus, plays a central role in the initiation of a non-alcoholic fatty liver disease-like lesion. These ndings indicate that periodontitis has an eect not only on diabetes mellitus and coronary heart diseases but also on liver disease. However, in the rat model of periodontal disease, serum levels of ALT were not increased (6), suggesting that periodontitis is not sucient to induce a functional disorder of the liver of rats in the absence of systemic diseases. Non-alcoholic fatty liver disease is recognized to be a hepatic manifestation of metabolic disorders (7) and is characterized by hepatocellular steatosis, apoptosis, inammatory inltration and brosis (8). It has been demonstrated that cholesterol overload (e.g. feeding a high-cholesterol diet) results in not only metabolic disorders but also hepatic brosis and inammation (9). Day & James (10) have suggested a two-hit theory in which the development of non-alcoholic fatty liver disease requires an additional etiological factor as well as steatosis. In their theory, oxidative stress is considered the most likely factor as the second hit, a key event for disease progression (10,11). Although periodontal inammation alone does not

Material and methods

Animals

Twenty-four male Wistar rats (8 weeks old) were used in this study. The animals were housed in steel cages in controlled conditions of 12 h light 12 h dark cycles, 50% humidity and 2225C. All experimental procedures were approved by the Animal Research Control Committee of Okayama University Dental School.
Experimental design

Animals were randomly divided into four groups of six rats each. During the experimental period, the rst two groups were fed with a normal diet for 8 weeks, and they received topical application of pyrogen-free water (control group) or 25 lg/lL Escherichia coli LPS and 2.25 U/lL Streptomyces griseus proteases (periodontitis group) to the gingival sulcus for 4 weeks prior to the end of the experimental period (14). The remaining two groups were fed a diet containing 1% cholesterol (w/w) and 0.5% cholic acid (w/w; Oriental Yeast Co., Tokyo, Japan) for 8 weeks, and they received topical application of pyrogen-free water (cholesterol group) or LPS and proteases (combination group) for 4 weeks prior to the end of the experimental period. Lipopolysaccharide

At the end of the experimental period, blood samples (2 mL) were collected directly from the heart of 24-h-fasted animals under general anesthesia with diethyl ether. Blood was allowed to clot at room temperature for 1 h, and serum was separated by centrifugation at 1500g for 15 min. Levels of serum total cholesterol and triglycerides were evaluated with the use of an enzymatic commercial kit (Cholesterol E-test Wako; Wako Pure Chemical Industries, Osaka, Japan; 15). The activities of serum AST and ALT were analyzed with a commercially available assay kit (Wako Pure Chemical Industries). Tumor necrosis factor-a (TNF-a) concentrations in serum were determined with a rat TNF-a enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Camarillo, CA, USA). Serum levels of C-reactive protein (CRP) were quantied by a highly sensitive ELISA (rat C-reactive protein ELISA test kit; Life Diagnostics, Inc., West Chester, PA, USA).
Measurements of serum oxidative stress

In order to measure serum oxidative stress, the N,N-diethylparaphenylendiamine reactive oxygen metabolites (d-ROMs) test was performed using the free radical elective evaluator (Diacron, Grosseto, Italy), according to the analysis procedures (16). A 20 lL serum sample and 1 mL of buered solution (N,N-diethylparaphenylendiamine, R2 reagent of the

Periodontitis, cholesterol and liver kit, pH 4.8) were gently mixed in a cuvette, and then 10 lL of chromogenic substrate (R1 reagent of the kit) was added to it. After mixing, the cuvette was immediately incubated in the thermostatic block of the analyzer for 5 min at 37C; 505 nm absorbance was then recorded. The measurement unit was expressed as the Carratelli unit (CARR U). It has been established that 1 CARR U corresponds to 0.08 mg/dL H2O2.
Measurement of serum lipid peroxides

131

Since the formation of hexanoyl-lysine (HEL) in lipid hydroperoxide-modied proteins, including oxidatively modied low-density lipoprotein, was reported as an initial marker for lipid peroxide (17), serum levels of HEL were assessed in triplicate using an HEL ELISA kit (Japan Institute for the Control of Aging, Shizuoka, Japan; 18).
Histological evaluation

at a magnication of 200 (6). The number of polymorphonuclear leukocytes (PMNs) and blood vessels in two standard areas (0.05 mm 0.05 mm each) of the connective tissue subjacent to the junctional epithelium was determined at a magnication of 400 (6). Three sections were selected from each rat and analyzed. Liver pathology was scored as described in a previous study (17), as follows: steatosis (the percentage of liver cells containing fat), <25% = 1+, <50% = 2+, <75% = 3+ and >75% = 4+; and inammation and necrosis, one focus per low-power eld = 1+, two or more = 2+.
Measurements of hepatic levels for 8hydroxydeoxyguanosine

periodontitis or combination groups; and the cholesterol or periodontitis vs. the combination groups) were made by the MannWhitney U-test. Since ve pairwise comparisons were performed, the statistically signicant level was set to 0.01 according to Bonferronis correction. All calculations were performed using a statistical software package (SPSS 15.0J for Windows; SPSS Japan, Tokyo, Japan).

Results
There were no signicant dierences among the four groups in terms of food consumption and body weight during the experimental period. Serum levels of total cholesterol, AST, ALT, CRP and HEL in the cholesterol group were signicantly higher than those in the control group (Table 1). For the periodontitis group, values of CRP, d-ROMs and HEL were signicantly higher than those for the control group. All the serum parameters except triglycerides in the combination group were signicantly higher than those in the control group. The level of serum TNF-a in the combination group was higher than those in the cholesterol and periodontitis groups. Linear distances between the CEJ and the most apical portion of the junctional epithelium and between the

The animals were killed at the end of the experimental period, under deep anesthesia with diethyl ether and exsanguination. The maxillary molar regions and liver samples were resected from each rat and immediately xed in 4% paraformaldehyde in 0.1 mol/L phosphate buer (pH 7.4) for 1 day. Periodontal tissues were further subjected to decalcication with 10% tetrasodium-EDTA aqueous solution (pH 7.4) for 2 weeks at 4C. The decalcied periodontal tissue samples and the formalin-xed liver tissue samples were embedded in paran and stained with hematoxylin and eosin. A single examiner, blind to the treatment assignment, performed the following histometric analyses using a light microscope. Periodontal sections stained with hematoxylin and eosin were used to evaluate the degree of periodontitis. The distances between the cemento-enamel junction (CEJ) and the alveolar bone crest (level of alveolar bone), and between the CEJ and the most apical portion of the junctional epithelium (degree of rete ridge elongation of junctional epithelium) were measured with a microgrid

Mitochondrial DNA was isolated from rat liver with a DNA extraction kit (Wako Pure Chemical Industries). Isolated mitochondrial DNA was analyzed by a competitive ELISA method with the use of an 8-hydroxydeoxyguanosine (8-OHdG) check kit (Japan Institute for the Control of Aging; 18).
Statistical analysis

Data are presented as means SD. Comparisons between the groups of rats (the control vs. the cholesterol,
Table 1. Results of serum parameters Control group (n = 6) Total cholesterol (mg/dL) Triglycerides (mg/dL) Aspartate aminotransferase (i.u./L) Alanine aminotransferase (i.u./L) Ratio of aspartate aminotransferase to alanine aminotransferase Tumor necrosis factor-a (pg/mL) C-reactive protein (mg/mL) N,N-diethylparaphenylendiamine reactive oxygen metabolites (CARR U) Hexanoyl-lysine (nmol/L) 56 9 22 8 106 22 21 4 5.3 1.6

Cholesterol group (n = 6) 146 17* 18 8 192 46* 42 19* 4.9 1.1

Periodontitis group (n = 6) 66 10 24 10 92 20 19 3 4.8 0.8

Combination group (n = 6) 140 55* 24 9 205 99 * 101 118* 2.9 1.1*

6.0 2.3 0.8 0.3 294 30

9.3 3.4 2.2 0.9* 443 184

6.0 2.0 2.0 0.6* 411 45*

18.7 5.4* 2.2 0.5* 697 203*

6.7 0.7

8.6 0.8*

11.1 3.0*

12.9 6.1*

Values are means SD. *p < 0.01 compared with the control group, using MannWhitney U-test. p < 0.01 compared with the cholesterol group, using MannWhitney U-test. p < 0.01 compared with the periodontitis group, using MannWhitney U-test.

132

Yamamoto et al.

Table 2. Histological analysis of rat periodontal tissue Control group (n = 6) Linear distance between the CEJ and apical portion of the junctional epithelium (lm) Linear distance between the CEJ and alveolar bone crest (lm) Polymorphonuclear leukocyte density (number per 0.05 mm 0.05 mm eld) Blood vessel density (number per 0.05 mm 0.05 mm eld) 00 560 61 1.4 0.5 1.3 0.3 Cholesterol group (n = 6) 50 21* 720 56* 1.9 0.3 2.2 0.3* Periodontitis group (n = 6) 154 26* 686 108 2.3 0.2* 2.1 0.2* Combination group (n = 6) 182 25* 837 152* 2.6 0.4* 2.2 0.4*

Values are means SD. *p < 0.01 compared with the control group, using MannWhitney U-test. p < 0.01 compared with the cholesterol group, using MannWhitney U-test.

CEJ and alveolar bone crest and blood vessel density in the cholesterol group were larger than those in the control group (Table 2). Linear distance between the CEJ and the most apical portion of the junctional epithelium and densities of PMNs and blood vessels in the periodontitis group were larger than those in the control group. Moreover, all four histological parameters for the combination group were larger than those in the control group. In the control group, few pathological changes were observed in the liver

(Fig. 1A). The cholesterol group showed moderate hepatic steatosis (Fig. 1B). In the periodontitis group, hepatocytes with small fatty droplets and scattered foci of inammatory cell inltration were observed (Fig. 1C). The degree of hepatic steatosis and inammation in the combination group was greater than those in the other groups (Fig. 1D). The cholesterol group showed higher steatosis and inammation scores for the liver than the control group (Table 3). The inammation score in the periodontitis

group was higher than that in the control group. Furthermore, the steatosis and inammation scores in the combination group were higher than those in the cholesterol group. The level of mitochondrial 8OHdG in the liver was higher in the cholesterol and periodontitis groups than in the control group (Fig. 2). The hepatic level of mitochondrial 8-OHdG in the combination group was higher than those in both the cholesterol and the periodontitis groups.

Discussion
Feeding a high-cholesterol diet increased serum levels of AST and ALT and scores of inammation and steatosis in the liver. In addition, a combination of high-cholesterol diet and topical application of a combination of LPS and proteases reduced the ratio of AST to ALT in serum and increased the 8-OHdG concentration in liver tissue. A decrease in the ratio of AST to ALT in serum and increase in 8OHdG concentration in liver indicate hepatic impairment (5) and oxidative DNA damage (19), respectively. Since additive eects were evident, topical application of a combination of LPS and proteases might contribute to exacerbate impairment and oxidative damage in the liver. Topical application of a combination of LPS and proteases to the gingival sulcus increased the steatosis in rats fed a high-cholesterol diet. This result agrees with a proposed theory in which endotoxemia resulting from intestinal bacterial overgrowth contributes to progression of steatosis to

Fig. 1. Liver from rats in the control, cholesterol, periodontitis and combination groups stained with hematoxylin and eosin. Few pathological changes were observed in the control group (A). The periodontitis group showed slight fatty change, characterized by small droplets in the hepatocytes (C). The fatty changes of cholesterol (B) and combination groups (D) were more severe than those of the periodontitis group (C). Focuses of hepatocytes containing fat vacuoles (arrows) are more common in the combination group (D) than any other groups. Scale bar represents 50 lm.

Periodontitis, cholesterol and liver

Table 3. Inammation, steatosis and necrosis scores in rat liver Control group (n = 6) Steatosis score <25% (1+) 2550% (2+) 5075% (3+) >75% (4+) Inammation score 0 1+ 2+ Necrosis score 0 1+ 2+ Cholesterol group (n = 6) Periodontitis group (n = 6) Combination group (n = 6)

133

6 0 0 0 6 0 0 6 0 0

0 1 5 0 0 4 2 3 3 0

3 2 1 0 0 6 0 4 2 0

0 0 4 2 0 0 6 2 3 1

Fig. 2. Hepatic levels of 8-OHdG in the control, cholesterol, periodontitis and combination groups. **p < 0.001, pairwise comparison using MannWhitney U-test.

non-alcoholic steatohepatitis, which represents an advanced stage of fatty liver disease (20). Other studies have indicated that hyperlipidemia frequently accompanies infectious disease (21). The results of the present study suggest that periodontal infection contributes to non-alcoholic fatty liver disease. If the relation is conrmed in human studies in the future, medical colleagues and patients with non-alcoholic fatty liver disease may consider periodontal health as an important factor for control of non-alcoholic fatty liver disease. Moreover, it may be recommended that patients with nonalcoholic fatty liver disease be referred

to dentists for checking and treatment of periodontal inammation. The exact pathological mechanisms of non-alcoholic fatty liver disease have not been fully elucidated; however, studies suggest that oxidative stress is involved in its pathogenesis. For instance, a placebo-controlled trial involving antioxidants (vitamin E and vitamin C treatment) showed an improvement of brosis in patients with non-alcoholic fatty liver disease (22). It is also known that oxidative stress can progress liver injury from steatosis to steatohepatitis mainly by three mechanisms: lipid peroxidation, cytokine induction and Fas ligand induction (23). In the present study, lipid peroxidation and oxidative stress might be responsible for liver injury in the combination group because serum levels of HEL and d-ROMs and liver levels of 8-OHdG were elevated. The results are consistent with studies in which parameters of oxidative stress, such as malonyldialdehyde (24), 4-hydroxynonenal (24) and thioredoxin (25) in blood and 4-hydroxynonenal and 8-OHdG in liver (26), were elevated in patients with non-alcoholic fatty liver disease. Recent studies demonstrated that a signicantly higher level of blood markers of oxidative stress was observed in chronic periodontitis patients than in periodontally healthy subjects (27,28). These ndings suggest that periodontal inammation increases the circulating markers of oxidative stress

in chronic periodontitis patients, but it is unclear how such conditions could be detrimental to general health. The present study supports the concept that increased circulating markers of oxidative stress following periodontal inammation could augment liver injury. In patients with hepatic steatosis, periodontal inammation may be a contributing factor for progression of non-alcoholic fatty liver disease. In such cases, evaluation of liver enzymes and oxidative stress in the clinic is recommended. Furthermore, the use of anti-oxidants (e.g. vitamin C treatment) in addition to periodontal treatment may be eective in preventing progression of non-alcoholic fatty liver disease in periodontitis patients. Other mechanisms might also be involved in enhancing steatosis and inammation in the liver induced by topical application of LPS and proteases. Topically applied LPS might directly aect liver cells through the systemic circulation. An increase in circulating LPS has been reported in our previous study using the same rat model of periodontal inammation (6). An increased number of blood vessels and the extension of blood vessels were found in the gingiva of the periodontitis and combination groups. These microvascular changes would enable entry of LPS from the gingival connective tissue into the systemic circulation. In fact, LPS applied into the gingival sulcus is transferred to blood vessels 2 h after application (29). Studies have shown that LPS elicits a wide variety of host defense responses to severe tissue injury, including liver injury in many models (30). Inammatory cytokines, such as TNF-a, which are produced in periodontal inammation, might directly aect liver cells. Steatosis is associated with increased TNF-a (20), the level of which was elevated in the serum of rats fed a high-cholesterol diet and with topical application of a combination of LPS and proteases in this study. Our previous study, using the same rat model and the same study design, showed that high dietary cholesterol could initiate and augment periodontal inammation (15), and the results were conrmed by the present study. In

134

Yamamoto et al. dontal inammation, a high-cholesterol diet and hepatic injury. In conclusion, local application of LPS and proteases to the gingival sulcus augments impairment and oxidative damage of the liver induced by feeding rats with a high-cholesterol diet.
plasma 8-OHdG level in rat periodontitis. Arch Oral Biol 2008;53:324329. Lu LS, Wu CC, Hung LM et al. Apocynin alleviated hepatic oxidative burden and reduced liver injury in hypercholesterolemia. Liver Int 2007;27:529537. Ekuni D, Yamamoto T, Yamanaka R, Tachibana K, Watanabe T. Proteases augment the effects of lipopolysaccharide in rat gingiva. J Periodontal Res 2003; 38:591596. Tomofuji T, Kusano H, Azuma T, Ekuni D, Yamamoto T, Watanabe T. Effects of a high-cholesterol diet on cell behavior in rat periodontitis. J Dent Res 2005;84:752 756. Komatsu F, Kagawa Y, Sakuma M et al. Investigation of oxidative stress and dietary habits in Mongolian people, compared to Japanese people. Nutr Metab (Lond) 2006;7:321. Uesugi T, Froh M, Arteel GE et al. Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice. J Immunol 2002;168:29632969. Kang MH, Naito M, Tsujihara N, Osawa T. Sesamolin inhibits lipid peroxidation in rat liver and kidney. J Nutr 1998; 128: 10181022. Kasai H. Chemistry-based studies on oxidative DNA damage: formation, repair, and mutagenesis. Free Radic Biol Med 2002;33:450456. Solga SF, Diehl A. Non-alcoholic fatty liver disease: lumen-liver interactions and possible role for probiotics. J Hepatol 2003;38:681687. Feingold KR, Staprans I, Memon RA et al. Endotoxin rapidly induces changes in lipid metabolism that produce hypertriglyceridemia: low doses stimulate hepatic triglyceride production while high doses inhibit clearance. J Lipid Res 1992;33:17651776. Harrison SA, Torgerson S, Hayashi P, Ward J, Schenker S. Vitamin E and vitamin C treatment improves fibrosis in patients with nonalcoholic steatohepatitis. Am J Gastroenterol 2003;98:24852490. Duvnjak M, Lerotic I, Barsic N, Tomasic V, Virovic JukicL, Velagic V. Pathogenesis and management issues for nonalcoholic fatty liver disease. World J Gastroenterol 2007;13:45394550. Loguercio C, De Girolamo V, de Sio I et al. Non-alcoholic fatty liver disease in an area of southern Italy: main clinical, histological, and pathophysiological aspects. J Hepatol 2001;35:568574. Sumida Y, Nakashima T, Yoh T et al. Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease. J Hepatol 2003; 38:3238. Seki S, Kitada T, Yamada T, Sakaguchi H, Nakatani K, Wakasa K. In situ detec-

another study, a high-cholesterol diet induced oxidative damage in the periodontium (31). Moreover, the present study showed that periodontal inammation augmented steatosis, inammation and oxidative damage in the liver. These results suggest that oxidative damage of both periodontium and liver was induced additionally by a high-cholesterol diet and periodontitis. For example, cholesterol overdose induces and/or exacerbates periodontal inammation, the periodontal inammation may induce local production of reactive oxygen species and cytokines, and the reactive oxygen species and cytokines may enter the systemic circulation and directly aect the liver. The degree of liver injury, i.e. scores of inammation, steatosis and necrosis of the liver, in the periodontitis group was less severe than those observed in the rat model of periodontal disease in our previous study (6). The dierence might be ascribed to the period of application of a combination of LPS and proteases to the gingival sulcus: 4 weeks in the present study, compared with 8 weeks in the previous study. Lipopolysaccharides from E. coli and proteases from S. griseus were applied to rat gingival sulcus in the present study. These bacterial species are not generally considered to be periodontal pathogens. Although both E. coli LPS and LPS from periodontopathic bacteria such as Porphyromonas gingivalis have similar eects on the induction of oxidative stress in gingival cells (32), a recent study suggested that E. coli LPS, but not P. gingivalis LPS induced inammatory responses in the heart/aorta (33). This is the potential limitation of this study. However, the application of E. coli LPS and S. griseus protease provide a rat periodontal disease model with high repeatability, and the use of commercial products ensures more uniform experimental conditions than with custom-made products (14,34). In addition, the present investigation was not a longitudinal study, and changes in parameters were not evaluated over time. Longitudinal studies will be needed to examine the causal relationships among perio-

13.

14.

Acknowledgements
This study was supported by a Grantin-Aid for Scientic Research (18592277) from the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.
15.

16.

References
1. Williams RC. Periodontal disease. N Engl J Med 1990;322:373382. 2. Mealey BL, Oates TW. American Academy of Periodontology. Diabetes mellitus and periodontal diseases. J Periodontol 2006;77:12891303. 3. Cutler CW, Iacopino AM. Periodontal disease: links with serum lipid/triglyceride levels? Review and new data J Int Acad Periodontol 2003;5:4751. 4. Morrison HI, Ellison LF, Taylor GW. Periodontal disease and risk of fatal coronary heart and cerebrovascular diseases. J Cardiovasc Risk 1999;6:711. 5. Saito T, Shimazaki Y, Koga T, Tsuzuki M, Ohshima A. Relationship between periodontitis and hepatic condition in Japanese women. J Int Acad Periodontol 2006;8:8995. 6. Tomofuji T, Ekuni D, Yamanaka R et al. Chronic administration of lipopolysaccharide and proteases induces periodontal inflammation and hepatic steatosis in rats. J Periodontol 2007;78:19992006. 7. Marchesini G, Brizi M, Bianchi G et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 2001;50:18441850. 8. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Non-alcoholic fatty liver disease: a spectrum of clinical and pathological study. Gastroenterology 1999;116:14131419. 9. Jeong WI, Jeong DH, Do SH et al. Mild hepatic fibrosis in cholesterol and sodium cholate diet-fed rats. J Vet Med Sci 2005;67:235242. 10. Day CP, James OF. Steatohepatitis: a tale of two hits? Gastroenterology 1998; 114: 842845. 11. Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest 2004;114:147152. 12. Ekuni D, Tomofuji T, Tamaki N et al. Mechanical stimulation of gingiva reduces 17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Periodontitis, cholesterol and liver

tion of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases. J Hepatol 2002;37:5662. 27. Akaln FA, Baltacoglu E, Alver A, Kar abulut E. Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis. J Clin Periodontol 2007;34:558565. 28. Baltacoglu E, Akaln FA, Alver A, Deger O, Karabulut E. Protein carbonyl levels in serum and gingival crevicular fluid in patients with chronic periodontitis. Arch Oral Biol 2008;53:716722. 29. Schwartz J, Stinson FL, Parker RB. The passage of tritiated bacterial endotoxin across intact gingival crevicular epithelium. J Periodontol 1972;43:270276. 30. Zhou Z, Wang L, Song Z et al. Abrogation of nuclear factor-jB activation is involved in zinc inhibition of lipopolysaccharide-induced tumor necrosis factor-a production and liver injury. Am J Pathol 2004; 164:15471556. 31. Tomofuji T, Azuma T, Kusano H et al. Oxidative damage of periodontal tissue in the rat periodontitis model: effects of a high-cholesterol diet. FEBS Lett 2006; 580:36013604. 32. Kim do Y, Jun JH, Lee HL et al. N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2

135

production in gingival fibroblasts. Arch Pharm Res 2007;30:12831292. 33. Liu R, Desta T, Raptis M, Darveau RP, Graves DT. P. gingivalis and E. coli lipopolysaccharides exhibit different systemic but similar local induction of inflammatory markers. J Periodontol 2008;79:1241 1247. 34. Ekuni D, Tomofuji T, Yamanaka R, Tachibana K, Yamamoto T, Watanabe T. Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases. J Periodontol 2005;76:4348.